ABSTRACT Hormonal therapy remains a mainstay of systemic treatment for the 70% of breast cancers that express the estrogen receptor (ER), but responses vary and intrinsic and acquired resistance are major clinical challenges. Recently, ligand-binding domain mutations in the estrogen receptor (ER) gene (ESR1) were found to occur and to mediate hormonal therapy resistance in approximately 35% of ER-positive (ER+) breast cancer patients. In the remaining two-thirds of cases, the molecular basis of hormonal therapy resistance is largely unknown. In preliminary data, based on targeted sequencing of 1756 breast cancer patients with recurrent or metastatic disease, we identified enrichment for alterations in diverse effectors of MAP kinase signaling (ERBB2 mutation, EGFR amplification, NF1 mutation, and others) in ESR1-wildtype breast cancers collected after disease progression on anti-estrogen therapy. This proposal is based on the hypothesis that recent advances in sequencing methodology and analysis of cell-free DNA (cfDNA) can prospectively identify molecular alterations that confer resistance to hormonal therapy in patients with breast cancer and that co-targeting such alterations alongside ER could prevent or delay the emergence of drug-resistant clones. Three specific aims are proposed. In Aim 1, paired tumor samples and cfDNA collected from breast cancer patients before and during treatment and at the time of disease progression on hormonal therapy will be used to define the landscape and timing of molecular changes that arise under the selective pressure of therapy. Our preliminary data indicate that ERBB2 mutation is a mechanism of both intrinsic and acquired resistance to hormonal therapy. We have also found that the HER kinase inhibitor neratinib can induce profound therapeutic responses that are limited by the induction of ER activity and consequent emergence of drug resistance. Thus, in Aim 2, we will use paired pretreatment and disease-progression tumor biopsies, cfDNA collected at regular intervals during treatment with neratinib alone or with the ER degrader fulvestrant, and engineered isogenic models and patient-derived xenografts to identify and validate mechanisms of resistance to these therapeutic agents. Finally, our genomic analysis identified RAS/ERK pathway alterations in ~10% of tumors collected following disease progression on hormonal therapy. In Aim 3, we will explore the functional significance of this finding and seek to develop therapeutic strategies for overcoming hormonal therapy resistance in this molecularly defined population. The long-term objective of this project is to develop therapeutic strategies for ER+ breast cancer guided by real-time, non-invasive molecular monitoring of tumor evolution. The work proposed will also generate valuable preclinical models of HER2-mutant and NF1-mutant ER+ breast cancer that can be used in future studies to assess promising novel therapeutic approaches for these molecularly defined populations.